|
| Status |
Public on Apr 10, 2012 |
| Title |
hour 72 post infection, 3 |
| Sample type |
RNA |
| |
|
| Source name |
Total RNA from infected A549 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549 cells
infection: H1N1 infection
time [post-infection]: hour 72
|
| Treatment protocol |
A549 cells were infected with the H1N1 virus at a multiplicity of infection (MOI) of 0.1. Infection with S-OIV 2009 H1N1 was performed in the presence of 1 μg/ml TPCK-trypsin. Cell supernatant and RNA were collected at 0, 4, 8, 24, 48, and 72 hours post-infection (hpi). The 0-hour time-point corresponds to samples collected immediately after the 1-hour virus incubation with additional time-points numbered with regard to the end of viral incubation. Mock-infected A549 cells were propagated for each experiment with samples collected at the 72-hour time-point.
|
| Growth protocol |
All experiments with live influenza virus were performed at the National Center for Foreign Animal Diseases under biosafety level 3 (BSL3+) conditions. A/Mexico/InDRE4487/2009 (H1N1) stocks were propagated on MDCK (Madin Darby canine kidney) cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the miRvana miRNA isolation kit (Ambion) following the instructions of the supplier.
|
| Label |
biotin
|
| Label protocol |
After a preincubation with 1× Klenow buffer, the enzymatic elongation was done with 0.25 U/μl Klenow Fragment (3′–5′ exo-) in a 1× buffer with biotinylated nucleotide (biotin-11-dATP, biotin-11-dCTP, biotin-11-dGTP or biotin-11-dUTP) at 37°C. The elongation was stopped using 6× SSPE buffer. Labeling of nucleotides utilized biotin with streptavidin–phwcoerythrin (SAPE) and a signal amplification step using biotinylated antistreptavidin antibodies and a second incubation with SAPE.
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| |
|
| Hybridization protocol |
As per the manufacturers instructions, hybridizations were performed overnight in the Febit GRTA(16 hours) at 42°C.
|
| Scan protocol |
Slides were scanned using a Geniom Real Time Analyzer (febit biomed gmbh). Wizard software on standard Microsoft Windows Workstation PC.
|
| Description |
Raw data file: run3.txt
See raw data file 'Array ID' column: 8
|
| Data processing |
For each array, the signal intensities for all miRNAs were extracted from the raw data file. As each miRNA was spotted in seven replicates, we obtained seven intensity values for each miRNA. The raw data files were used to obtain the median signal intensity of all features for each array. The values were background-corrected using the median for each array of blank controls. One intensity value was retained for each miRNA by calculating the median for all the corresponding replicates. Variance stabilizing normalization (VSN) was applied to normalize the data across different arrays. The H1N1 RNA in the uninfected samples in chip 1 and the hour 4 samples in chip 2 were found to be degraded during the mRNA profiling; therefore, they were excluded from the statistical analysis. Analyses were carried out using R.
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| |
|
| Submission date |
Mar 13, 2012 |
| Last update date |
Apr 10, 2012 |
| Contact name |
Victoria Svinti |
| Organization name |
University of British Columbia
|
| Street address |
2350 Health Sciences Mall
|
| City |
Vancouver |
| ZIP/Postal code |
V6T 1Z3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL15271 |
| Series (2) |
| GSE36461 |
MiRNA profiling during infection with H1N1 influenza A virus (A/Mexico/InDRE4487/H1N1/2009) |
| GSE36555 |
Host-influenza A virus(infA) interactions |
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